Pseudomonas syringae harpins, hopptop and hoppmahpto, and their uses

ABSTRACT

The present invention is directed to isolated proteins or polypeptides which elicit a hypersensitive response in plants, as well as isolated DNA molecules which encode the hypersensitive response eliciting proteins or polypeptides. These isolated proteins or polypeptides and the isolated DNA molecules can be used to impart disease resistance, stress resistance, and enhanced growth to plants or plants grown from treated seeds, to control insects on plants or plants grown from treated plant seeds, to impart post-harvest disease or desiccation resistance in fruits or vegetables, to impart enhanced longevity of fruit or vegetable ripeness, to impart desiccation resistance to cuttings of ornamental plants, and/or promote early flowering of ornamental plants, either by topical application of the proteins or polypeptides or transgenic expression in recombinant plants or plant seeds.

This application is entitled to priority benefit of U.S. ProvisionalPatent Application Ser. No. 60/356,408, filed Feb. 12, 2002, and U.S.Provisional Patent Application Ser. No. 60/380,185, filed May 10, 2002,each of which is hereby incorporated by reference in its entirety.

This invention was developed with government funding under NationalScience Foundation Grant Nos. MCB 9631530, MCB-9982646, and DBI-0077622.The U.S. Government may retain certain rights.

FIELD OF THE INVENTION

The present invention relates to new hypersensitive response elicitorproteins or polypeptides of Pseudomonas syringae and their uses.

BACKGROUND OF THE INVENTION

Interactions between bacterial pathogens and their plant hosts generallyfall into two categories: (1) compatible (pathogen-host), leading tointercellular bacterial growth, symptom development, and diseasedevelopment in the host plant; and (2) incompatible (pathogen-nonhost),resulting in the hypersensitive response, a particular type ofincompatible interaction occurring, without progressive diseasesymptoms. During compatible interactions on host plants, bacterialpopulations increase dramatically and progressive symptoms occur. Duringincompatible interactions, bacterial populations do not increase, andprogressive symptoms do not occur.

The hypersensitive response (“HR”) is a rapid, localized necrosis thatis associated with the active defense of plants against many pathogens(Horsfall et al., eds., Plant Disease: An Advanced Treatise, Vol. 5, pp.201-224, New York, N.Y.: Academic Press (1980); Mount et al., eds.,Phytopathogenic Prokaryotes, Vol. 2, pp. 149-177, New York, N.Y.:Academic Press (1982)). The hypersensitive response elicited by bacteriais readily observed as a tissue collapse if high concentrations (≧10⁷cells/ml) of a limited host-range pathogen like Pseudomonas syringae orErwinia amylovora are infiltrated into the leaves of nonhost plants(necrosis occurs only in isolated plant cells at lower levels ofinoculum) (Klement, Nature 199:299-300 (1963); Klement et al.,Phytopathology 54:474-477 (1963); Turner et al., Phytopathology64:885-890 (1974); Mount et al., eds., Phytopathogenic Prokaryotes, Vol.2., pp. 149-177, New York, N.Y.: Academic Press (1982)). The capacitiesto elicit the hypersensitive response in a nonhost and be pathogenic ina host appear linked. As noted by Mount et al., eds., PhytopathogenicProkaryotes, Vol. 2., pp. 149-177, New York, N.Y.: Academic Press(1982), these pathogens also cause physiologically similar, albeitdelayed, necroses in their interactions with compatible hosts.Furthermore, the ability to produce the hypersensitive response orpathogenesis is dependent on a common set of genes, denoted hrp(Lindgren et al., J. Bacteriol. 168:512-22 (1986); Willis et al., Mol.Plant-Microbe Interact. 4:132-138 (1991)). Consequently, thehypersensitive response may hold clues to both the nature of plantdefense and the basis for bacterial pathogenicity.

The hrp genes are widespread in gram-negative plant pathogens, wherethey are clustered, conserved, and in some cases interchangeable (Williset al., Mol. Plant-Microbe Interact. 4:132-138 (1991); Dangl, ed.,Current Topics in Microbiology and Immunology: Bacterial Pathogenesis ofPlants and Animals—Molecular and Cellular Mechanisms, pp. 79-98, Berlin:Springer-Verlag (1994)). Several hrp genes encode components of aprotein secretion pathway similar to one used by Yersinia, Shigella, andSalmonella spp. to secrete proteins essential in animal diseases (VanGijsegem et al., Trends Microbiol. 1:175-180(1993)). In E. amylovora, P.syringae, and P. solanacearum, hrp genes have been shown to control theproduction and secretion protein elicitors of the hypersensitiveresponse (He et al., Cell 73:1255-1266 (1993); Wei et al., J. Bacteriol.175:7958-7967 (1993); Arlat et al., EMBO J. 13:543-553 (1994)).Hypersensitive response elicitor proteins, designated harpins, areproteins found in phytopathogens containing a type III secretion systemand are typically glycine-rich, acidic, cysteine-lacking, heat stableproteins (He et al., Cell 73: 1255-1266 (1993).

The first of these proteins was discovered in E. amylovora Ea321, abacterium that causes fire blight of rosaceous plants, and wasdesignated harpin (Wei et al., Science 257:85-88 (1992)). Mutations inthe encoding hrpN gene revealed that harpin is required for E. amylovorato elicit a hypersensitive response in nonhost tobacco leaves and incitedisease symptoms in highly susceptible pear fruit. The P. solanacearumGMI1000 PopA1 protein has similar physical properties and also elicitsthe hypersensitive response in leaves of tobacco, which is not a host ofthat strain (Arlat et al., EMBO J. 13:543-53 (1994)). However, P.solanacearum popA mutants still elicit the hypersensitive response intobacco and incite disease in tomato. Thus, the role of theseglycine-rich hypersensitive response elicitors can vary widely amonggram-negative plant pathogens.

Other plant pathogenic hypersensitive response elicitors have beenisolated, cloned, and sequenced from various organisms, including: HrpWfrom Erwinia amylovora (Kim et al., J. Bacteriol. 180(19):5203-5210(1998)); HrpN from Erwinia chrysanthemi (Bauer et al., MPMI 8(4): 484-91(1995)); HrpN from Erwinia carotovora (Cui et al., MPMI 9(7): 565-73(1996)); HrpN from Erwinia stewartii (Ahmad et al., 8th Int'l. Cong.Molec. Plant-Microb. Inter. Jul. 14-19, 1996 and Ahmad et al., Ann. Mtg.Am. Phytopath. Soc. Jul. 27-31, 1996); hreX from Xanthomonas campestrisU.S. Patent Application Publ. No. 20020066122 to Wei et al.); HrpZ fromPseudomonas syringae pv. syringae (He et al., Cell 73:1255-1266 (1993);WO 94/26782 to Cornell Research Foundation, Inc.); and HrpW fromPseudomonas syringae pv. tomato (Charkowski et al., J. Bacteriol.180:5211-5217 (1998)).

In electron microscopy studies, both HrpW and HrpZ of Pseudomonassyringae are associated with the type III secretion system pilus (Jin etal., Science 294:2556-2558 (2001); Jin et al., Molecular Microbiology40:1129-1139 (2001)), suggesting that harpins work with the pilus tofacilitate protein delivery into the plant cell. A P. syringae straincontaining chromosomal deletions of hrpZ and hrpW has a reduced abilityto cause the HR on nonhost plants, but it retains normal virulence ontomato (Charkowski et al., J. Bacteriol. 180:5211-5217 (1998)). Thisphenotype indicates that it is likely that there are more harpins in thegenome.

The present invention is a further advance in the effort to identify,clone, and sequence hypersensitive response elicitor proteins orpolypeptides from plant pathogens.

SUMMARY OF THE INVENTION

The present invention is directed to isolated proteins or polypeptideswhich elicit a hypersensitive response in plants as well as isolated DNAmolecules which encode the hypersensitive response eliciting proteins orpolypeptides.

The hypersensitive response eliciting proteins or polypeptides can beused to impart disease resistance, stress resistance, and enhancedgrowth to plants or plants grown from treated seeds, to control insectson plants or plants grown from treated plant seeds, to impartpost-harvest disease or desiccation resistance in fruits or vegetables,to impart enhanced longevity of fruit or vegetable ripeness, to impartdesiccation resistance to cuttings of ornamental plants, and/or promoteearly flowering of ornamental plants. This involves applying thehypersensitive response elicitor protein or polypeptide in anon-infectious form to plants, plant seeds, cuttings removed fromplants, and/or fruits or vegetables removed from plants under conditionseffective to impart disease resistance, stress resistance, and enhancedgrowth to plants or plants grown from treated seeds, to control insectson plants or plants grown from treated plant seeds, to impartpost-harvest disease or desiccation resistance in fruits or vegetables,to impart enhanced longevity of fruit or vegetable ripeness, to impartdesiccation resistance to cuttings of ornamental plants, and/or promoteearly flowering of ornamental plants.

As an alternative to applying the hypersensitive response elicitorprotein or polypeptide to plants or plant seeds in order to impartdisease resistance, stress resistance, and enhanced growth to plants orplants grown from treated seeds, to control insects on plants or plantsgrown from treated plant seeds, to impart post-harvest disease ordesiccation resistance in fruits or vegetables, to impart enhancedlongevity of fruit or vegetable ripeness, to impart desiccationresistance to cuttings of ornamental plants, and/or promote earlyflowering of ornamental plants, transgenic plants or plant seeds can beutilized. When utilizing transgenic plants, this involves providing atransgenic plant transformed with a DNA molecule encoding ahypersensitive response elicitor protein or polypeptide and growing theplant under conditions effective to impart disease resistance, stressresistance, and enhanced growth to plants, to control insects on plants,to impart post-harvest disease or desiccation resistance in fruits orvegetables, to impart enhanced longevity of fruit or vegetable ripeness,to impart desiccation resistance to cuttings of transgenic ornamentalplants, and/or promote early flowering of transgenic ornamental plants.Alternatively, a transgenic plant seed transformed with the DNA moleculeencoding a hypersensitive response elicitor protein or polypeptide canbe provided and planted in soil. A plant is then propagated underconditions effective to impart disease resistance, stress resistance,and enhanced growth to plants grown from the transgenic seeds, tocontrol insects on plants grown from the transgenic plant seeds, toimpart post-harvest disease or desiccation resistance in fruits orvegetables, to impart enhanced longevity of fruit or vegetable ripeness,to impart desiccation resistance to cuttings of the transgenicornamental plants, and/or promote early flowering of the transgenicornamental plants.

The present invention is also directed to a composition including acarrier and a protein or polypeptide of the present invention whichelicits a hypersensitive response in plants. The composition may alsoinclude an additive such as fertilizer, insecticide, fungicide,nematacide, and mixtures of these additives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a comparison between the amino acid sequences ofHrpZ, HrpW, HopPtoP, and HopPmaH from Pseudomonas syringae. Harpinproteins were analyzed using BLAST, and the percent similarities shownwere determined using protein-protein BLAST. Like HrpZ and HrpW, twopreviously identified harpins (see He et al., Cell 73:1255-1266 (1993);Charkowski et al., J. Bacteriol. 180:5211-5217 (1998), each of which ishereby incorporated by reference in its entirety), HopPtoP andHopPmaH_(Pto) have N-terminal domains with no predicted function. TheC-terminal domains of HopPtoP and HopPmaH_(Pto) show homologies toprotein domains with enzymatic function. Lytic transglycosylasesnormally break down peptidoglycan in the bacterial cell wall, whilepectin and pectate lyases break down pectin in the plant cell wall.

FIG. 2 shows the results of protein infiltration onto leaf surfaces andthe resulting elicitation of a hypersensitive response (“HR”). HrpZ,HopPtoP, and HopPmaH_(Pto) were purified using a 6×His tag system, anddenatured at 100° C. for 10 minutes. Proteins were infiltrated intoNicotiana tabacum cv. Xanthi, and the hypersensitive response wasphotographed at 24 hours.

FIGS. 3A-B illustrate the results of a secretion assay on HopPtoP andHopPmaH_(Pto), respectively. HopPtoP and HopPmaH_(Pto) were tagged atthe C-terminus with the adenylate cyclase (CyaA) protein for detectionin subsequent Western analysis of the cell pellet (cell) and supernatant(SN). The plasmids were expressed in either P. syringae DC3000 or P.syringae DC3000 containing a non-functional type III secretion system(Δhrp/hrc). Both proteins are secreted by P. syringae DC3000 but not P.syringae DC3000 (Δhrp/hrc).

FIG. 4 is a graph depicting the results of a translocation assay onHopPtoP and HopPmaH_(Pto) relative to HrpW and HrpZ. Each harpin genewas fused with the adenylate cyclase (CyaA) reporter gene on aninducible plasmid, and expressed in either P. syringae DC3000 or P.syringae DC3000 containing a non-functional type III secretion system(Δhrp/hrc). CyaA is activated by the protein calmodulin, which is foundonly in eukaryotic cells, and produces cAMP from ATP. Increased cAMPlevels can be measured, thereby detecting levels of translocation. Thestrains containing the CyaA fusions were infiltrated into tomato cv.Moneymaker plants, and leaf discs were collected 8 hourspost-infiltration for analysis of cAMP levels. Both HopPtoP andHopPmaH_(Pto) are translocated into leaf tissues in the presence of atype III secretion system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated DNA molecule having thenucleotide sequence of SEQ ID NO: 1 as follows: atgaccatgg gtgtttcacctattcgtaac tcaaactccc tgccgatcga tttttcgtcg 60 ttgagcgcaa agagtggcgggcataacggg ctgggcagcg gagacaattc gactatcgac 120 ccgagtacgt tgttgttcggcaatcaaggg cagacgcagg tcaatttcgc tccgcccaac 180 agcacggact cctcaaccagcggtgtgaac gctgcgtcag gcaatacggc gtccggcctg 240 gtcgagcaaa tcatgagcctgctgaaacaa ttgatgcaga tgctgatgca aaacaacaat 300 gcttccggta accctcagactgattcgtca acgccaggcg tcggcagtgg caacagcgtc 360 gggagcggcg gtactggaagcagtctcgca ggcagtgacg gtggcgacga aacgtccggt 420 gtcggtaacg gcggtttaggcgacgcgggc agcacgccaa caacgagcgc ggccgatggt 480 gtgccctcgg atacttcactcacgggtagc ggtgggctgc atttgcctca acagcttgag 540 cagtatcgag gcgacattatggacgccgcc aaagccaccg gcgtgccgcc cagcgtgatc 600 gccgggcaga tatgggctgagtcgcgcggt cagttgaatg cggccaccac caatgtcaac 660 ggcaaggccg atgcgggcctgatgcaggtc aacgcagaca cgttcaagtc attgcagcag 720 caaaacccgg ggttgctgggcaacgacgtc aacgattcgc acaccaacat catggcgggc 780 gcgctctacc tgcgagaccagaacaaggag ttcggcgaca tgggggcagc acttcgcgca 840 tacaactccg ggcccgacaaggtcaataaa gccgacctca gcgacacggg aggcgtgggc 900 ggcagcagct acccggcggacgtactgaac ttcgcgaaaa tcatcgagag tgggcagggc 960 aatttacccg cttga 975

This DNA molecule of the present invention encodes a protein orpolypeptide having the amino acid sequence of SEQ ID NO: 2 as follows:Met Thr Met Gly Val Ser Pro Ile Arg Asn Ser Asn Ser Leu Pro Ile  1               5                  10                  25 Asp Phe SerSer Leu Ser Ala Lys Ser Gly Gly His Asn Gly Leu Gly             20                  25                  30 Ser Gly Asp AsnSer Thr Ile Asp Pro Ser Thr Leu Leu Phe Gly Asn         35                  40                  45 Gln Gly Gln Thr GlnVal Asn Phe Ala Pro Pro Asn Ser Thr Asp Ser     50                  55                  60 Ser Thr Ser Gly Val AsnAla Ala Ser Gly Asn Thr Ala Ser Gly Leu 65                  70                  75                  80 Val GluGln Ile Met Ser Leu Leu Lys Gln Leu Met Gln Met Leu Met                 85                  90                  95 Gln Asn AsnAsn Ala Ser Gly Asn Pro Gln Thr Asp Ser Ser Thr Pro            100                 105                 110 Gly Val Gly SerGly Asn Ser Val Gly Ser Gly Gly Thr Gly Ser Ser        115                 120                 125 Leu Ala Gly Ser AspGly Gly Asp Glu Thr Ser Gly Val Gly Asn Gly    130                 135                 140 Gly Leu Gly Asp Ala GlySer Thr Pro Thr Thr Ser Ala Ala Asp Gly145                 150                 155                 160 Val ProSer Asp Thr Ser Leu Thr Gly Ser Gly Gly Leu His Leu Pro                165                 170                 175 Gln Gln LeuGlu Gln Tyr Arg Gly Asp Ile Met Asp Ala Ala Lys Ala            180                 185                 190 Thr Gly Val ProPro Ser Val Ile Ala Gly Gln Ile Trp Ala Glu Ser        195                 200                 205 Arg Gly Gln Leu AsnAla Ala Thr Thr Asn Val Asn Gly Lys Ala Asp    210                 215                 220 Ala Gly Leu Met Gln ValAsn Ala Asp Thr Phe Lys Ser Leu Gln Gln225                 230                 235                 240 Gln AsnPro Gly Leu Leu Gly Asn Asp Val Asn Asp Ser His Thr Asn                245                 250                 255 Ile Met AlaGly Ala Leu Tyr Leu Arg Asp Gln Asn Lys Glu Phe Gly            260                 265                 270 Asp Met Gly AlaAla Leu Arg Ala Tyr Asn Ser Gly Pro Asp Lys Val        275                 280                 285 Asn Lys Ala Asp LeuSer Asp Thr Gly Gly Val Gly Gly Ser Ser Tyr    290                 295                 300 Pro Ala Asp Val Leu AsnPhe Ala Lys Ile Ile Glu Ser Gly Gln Gly305                 310                 315                 320 Asn LeuPro Ala

This protein or polypeptide (also referred to herein as “HopPtoP”) has apredicted molecular mass of about 32 kDa and an isoelectric point ofabout 4.13. Like other hypersensitive response elicitors, the aboveprotein is glycine rich (˜15%), lacks cysteine, is sensitive toproteases, is temperature stable, is secreted via a type III secretionsystem (where it appears to be targeted to the plant apoplast), and iscapable of inducing a hypersensitive response following infiltrationonto plant tissues of non-host plants. Like HrpW, HopPtoP has a striking2-domain structure with the N-terminal portion being “harpin-like” (aa1-189) and the second portion having homology to lytic transglycosylate(aa 190-324). The harpin domain of HopPtoP possess approximately 53%similarity and 34% identity to the corresponding harpin domain of HrpWfrom Pseudomonas syringae from DC3000. The lytic transglycosylate domainof HopPtoP shares about 54% similarity to a lytic transglycosylase ofMezorhizobium sp. Based on these similarities, HopPtoP is considered ahomolog of HrpW.

The present invention also relates to an isolated DNA molecule havingthe nucleotide sequence of SEQ ID NO: 3 as follows: atgaatacgatcaacagaaa catctacccc gtctccggga tttctgcgca ggatgcccct 60 gtacaaactgatcagctcca gccgcaaggc cagggcatca ggccggggca caatagcaac 120 ctgatcgacttcggactgat acagcaggcc aatggtccgc actcatcgct gaacacatcg 180 agctccagaattcagccgac tgacaccagc acatcctcaa acaggctggg gggtaatggc 240 gatcagttactgaacaaact cgtggaagcg atccgtaata tcctcaacaa cctgctctct 300 ctgctggaaggcaatcaaca ccagggctct tcgcctgcac agacccagcg tgaacagacg 360 ccgacgtccactcaatcgca cgcttcgcct tcctcgtcgt cttcatcttc gccgtcgaca 420 tcctcccagtcttcaccctc agtgccttca acgcctcagg gcaacgcaga aaaaccgttt 480 gtggtgcagagcgatcatcc ggcggaaaaa ccggtatcgc tgcagagaac ctcagagcca 540 acgtctgtgacgccgccaca aacaccaccg caggctgtcg agcgaaacag cattaccccg 600 gacaaggcaccggccaaacc cgaagcggta aagccggcag tggtcaacga cccggtgctg 660 ccgaaaacctcgatccctgc cgccgccaag cctgacagca cggtgaccgc cgcaaaacac 720 gcgacgcccgctgcccgtgg ccagggcgct gacatgtccg gcatgatcgg ttttgccaag 780 gaagccaataccaccggggg caacaacggc gaagtggtca ccgtgaacac ggttgccgac 840 ctcaagaagtacatggagga cgacaaagcc cgcaccgtca agctgggggC caacctgtct 900 gccgacagtaaagtgtcgat aaatttcggg gccaacaaaa ccctgctggg caccgataaa 960 ggcaacaccctgcacaacat ctatctggcc agcggcaaga ccgccagcaa cgacattttc 1020 cagaatctgaacttcaacca cgacgcccgt taccgtgaaa acggcgacat gcagatgttc 1080 atcagcagcggtcagaaata ctggatcgac cacatcaccg ctaccggaac caaggatcag 1140 aaccccaaaggtctggataa actgctctac gtgggcggca aggcagataa cgtcagcctg 1200 accaattcgaaattccagaa caacgagtat ggcgtgattc tcggtcagcc ggacgactcg 1260 gcagccgccaaagccgagta caagggctac ccacggatga caatcgccaa caacgtgttc 1320 agcaacctcgatgtccgcgg gcccggtctg tttcgtcagg gccaatttga cgtagttaac 1380 aactcgatcgacaaattcca cctcggtttc actgcgaccg ggaacgctac catcctgtcg 1440 caggccaactatttcagcaa cggtgtcgat gtttccaaca aggcaagtaa tagcggcgtg 1500 ctggatgactacggcgatgc gcacttcaaa gacatcggca gtaacgtcag tttcactcag 1560 aaatcgccggttaccgcctg gacaccgagc tacaaccggg acgtgaaaac agccgaagca 1620 gccagagcctatgacctggc caatgcgggt gcacaggtcg tgaaataa 1668

This DNA molecule of the present invention encodes a protein orpolypeptide having the amino acid sequence of SEQ ID NO: 4 as follows:Met Asn Thr Ile Asn Arg Asn Ile Tyr Pro Val Ser Gly Ile Ser Ala  1               5                  10                  25 Gln Asp AlaPro Val Gln Thr Asp Gln Leu Gln Pro Gln Gly Gln Gly             20                  25                  30 Ile Arg Pro GlyHis Asn Ser Asn Leu Ile Asp Phe Gly Leu Ile Gln         35                  40                  45 Gln Ala Asn Gly ProHis Ser Ser Leu Asn Thr Ser Ser Ser Arg Ile     50                  55                  60 Gln Pro Thr Asp Thr SerThr Ser Ser Asn Arg Leu Gly Gly Asn Gly 65                  70                  75                  80 Asp GlnLeu Leu Asn Lys Leu Val Glu Ala Ile Arg Asn Ile Leu Asn                 85                  90                  95 Asn Leu LeuSer Leu Leu Glu Gly Asn Gln His Gln Gly Ser Ser Pro            100                 105                 110 Ala Gln Thr GlnArg Glu Gln Thr Pro Thr Ser Thr Gln Ser His Ala        115                 120                 125 Ser Pro Ser Ser SerSer Ser Ser Ser Pro Ser Thr Ser Ser Gln Ser    130                 135                 140 Ser Pro Ser Val Pro SerThr Pro Gln Gly Asn Ala Glu Lys Pro Phe145                 150                 155                 160 Val ValGln Ser Asp His Pro Ala Glu Lys Pro Val Ser Leu Gln Arg                165                 170                 175 Thr Ser GlnPro Thr Ser Val Thr Pro Pro Gln Thr Pro Pro Gln Ala            180                 185                 190 Val Glu Arg AsnSer Ile Thr Pro Asp Lys Ala Pro Ala Lys Pro Glu        195                 200                 205 Ala Val Lys Pro AlaVal Val Asn Asp Pro Val Leu Pro Lys Thr Ser    210                 215                 220 Ile Pro Ala Ala Ala LysPro Asp Ser Thr Val Thr Ala Ala Lys His225                 230                 235                 240 Ala ThrPro Ala Ala Arg Gly Gln Gly Ala Asp Met Ser Gly Met Ile                245                 250                 255 Gly Phe AlaLys Glu Ala Asn Thr Thr Gly Gly Asn Asn Gly Glu Val            260                 265                 270 Val Thr Val AsnThr Val Ala Asp Leu Lys Lys Tyr Met Glu Asp Asp        275                 280                 285 Lys Ala Arg Thr ValLys Leu Gly Ala Asn Leu Ser Ala Asp Ser Lys    290                 295                 300 Val Ser Ile Asn Phe GlyAla Asn Lys Thr Leu Leu Gly Thr Asp Lys305                 310                 315                 320 Gly AsnThr Leu His Asn Ile Tyr Leu Ala Ser Gly Lys Thr Ala Ser                325                 330                 335 Asn Asp IlePhe Gln Asn Leu Asn Phe Asn His Asp Ala Arg Tyr Arg            340                 345                 350 Glu Asn Gly AspMet Gln Met Phe Ile Ser Ser Gly Gln Lys Tyr Trp        355                 360                 365 Ile Asp His Ile ThrAla Thr Gly Thr Lys Asp Gln Asn Pro Lys Gly    370                 375                 380 Leu Asp Lys Leu Leu TyrVal Gly Gly Lys Ala Asp Asn Val Ser Leu385                 390                 395                 400 Thr AsnSer Lys Phe Gln Asn Asn Glu Tyr Gly Val Ile Leu Gly Gln                405                 410                 415 Pro Asp AspSer Ala Ala Ala Lys Ala Glu Tyr Lys Gly Tyr Pro Arg            420                 425                 430 Met Thr Ile AlaAsn Asn Val Phe Ser Asn Leu Asp Val Arg Gly Pro        435                 440                 445 Gly Leu Phe Arg GlnGly Gln Phe Asp Val Val Asn Asn Ser Ile Asp    450                 455                 460 Lys Phe His Leu Gly PheThr Ala Thr Gly Asn Ala Thr Ile Leu Ser465                 470                 475                 480 Gln AlaAsn Tyr Phe Ser Asn Gly Val Asp Val Ser Asn Lys Ala Ser                485                 490                 495 Asn Ser GlyVal Leu Asp Asp Tyr Gly Asp Ala His Phe Lys Asp Ile            500                 505                 510 Gly Ser Asn ValSer Phe Thr Gln Lys Ser Pro Val Thr Ala Trp Thr        515                 520                 525 Pro Ser Tyr Asn ArgAsp Val Lys Thr Ala Glu Ala Ala Arg Ala Tyr    530                 535                 540 Asp Leu Ala Asn Ala GlyAla Gln Val Val Lys 545                 550                 555

This protein or polypeptide (also referred to herein as “HopPmaH_(Pto)”)has a predicted molecular mass of about 59 kDa and an isoelectric pointof about 7.65. Like other hypersensitive response elicitors, the aboveprotein is glycine rich (˜7.5%), lacks cysteine, is sensitive toproteases, is temperature stable, is secreted via a type III secretionsystem, and is capable of inducing a hypersensitive response followinginfiltration onto plant tissues of non-host plants. Like HrpW,HopPmaH_(Pto) has a striking 2-domain structure with the N-terminalportion (aa 1-279) being “harpin-like” and the second portion (aa280-555) having homology to pectin/pectate lyase. The pectate-lyasedomain is about 60% similar and about 43% identical to thepectate/pectin lyase from Bacillus subtilis (Accession No. AF027868,which is hereby incorporated by reference in its entirety). Within thelyase domain, HopPmaH_(Pto) is overall about 30% similar and 22%identical to the pectate/pectin lyase from B. subtilis.

Fragments of the above hypersensitive response elicitor polypeptides orproteins are encompassed by the present invention. Suitable fragmentscan include those portions of the harpin proteins that contain theharpin-like domain.

Suitable fragments can be produced by several means. In the firstapproach, subclones of the gene encoding the elicitor protein of thepresent invention are produced by conventional molecular geneticmanipulation by subcloning gene fragments. The subclones then areexpressed in vitro or in vivo in bacterial cells to yield a smallerprotein or peptide that can be tested for elicitor activity according tothe procedure described below, e.g., in Wei et al., Science 257:85-86(1992), which is hereby incorporated by reference in its entirety.

As an alternative approach, fragments of an elicitor protein can beproduced by digestion of a full-length elicitor protein with proteolyticenzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin.Different proteolytic enzymes are likely to cleave elicitor proteins atdifferent sites based on the amino acid sequence of the elicitorprotein. Some of the fragments that result from proteolysis may beactive elicitors of the hypersensitive response.

In another approach, based on knowledge of the primary structure of theprotein, fragments of the elicitor protein gene may be synthesized byusing the PCR technique together with specific sets of primers chosen torepresent particular portions of the protein. These then would be clonedinto an appropriate vector for increased expression of a truncatedpeptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for theelicitor being produced. Alternatively, subjecting a full lengthelicitor to high temperatures and pressures will produce fragments.These fragments can then be separated by conventional procedures (e.g.,chromatography, SDS-PAGE).

Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theproperties, secondary structure and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Also suitable as isolated nucleic acid molecules according to thepresent invention is a nucleic acid which has a nucleotide sequence thatis at least about 55% similar, preferably at least about 65% similar ormore preferably at least about 75% similar, to the nucleotide sequenceof SEQ ID NO: 1 or SEQ ID NO: 3 by basic BLAST using default parametersanalysis. Even more preferred is that such a nucleotide sequence have anucleic acid identity to the nucleotide sequence of SEQ ID NO: 1 or SEQID NO: 3, either by basic BLAST or ClustalW using default parametersanalysis, that is at least about 40%, more preferably at least about50%, and even more preferably at least about 60%. Higher percentages ofidentity and/or similarity are even more preferred, such as at leastabout 80% identity and/or at least about 85% similarity.

Also suitable as an isolated nucleic acid according to the presentinvention is an isolated nucleic acid molecule that hybridizes to thenucleotide sequence of SEQ ID NO: 1 (or its complement) or SEQ ID NO: 3(or its complement) under suitably stringent hybridization conditions.Exemplary stringent conditions include the use of a hybridization mediumor buffer that contains 5×SSC buffer at a temperature of about 42°-65°C., with hybridization being carried out for about 18-20 hours.Stringency can, of course, be increased by lowering the saltconcentration or increasing the temperature at which hybridizationoccurs. Thus, in another embodiment, the hybridization temperature isbetween about 52°-60° C. In yet another embodiment, the hybridization isbetween 55°-57° C. Another example of suitable high stringencyconditions is when hybridization is carried out at 65° C. for 20 hoursin a medium containing 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1%sodium dodecyl sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2%bovine serum albumin, 50 μm g/ml E. coli DNA. Wash conditions can beselected at varying stringency requirements, as long as the washconditions are suitable to remove non-specifically bound nucleic acidmolecules. Typically, when nucleic acid molecules of longer than about200 bases are used as probes in hybridization protocols (such asradiolabeled DNA molecules of SEQ ID NO: 1 or SEQ ID NO: 3), theimportance of the wash conditions is minimized. See Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Press, NY (1989), which is hereby incorporated by reference inits entirety. Thus, a series of increasingly stringent wash conditionscan be performed, beginning with lower stringency conditions andcontinuing until the stringency conditions of the hybridizationprocedure is achieved. The series of washes can be performed forsuitable time periods, typically anywhere from about 15 minutes up toabout 2 hours, although shorter or longer washes are certainlyeffective. However, any DNA molecules hybridizing to the DNA molecule ofSEQ ID NO: 1 (or its complement) or SEQ ID NO: 3 (or its complement)under such stringent conditions must not be identical to the nucleicacids encoding the HrpW hypersensitive response elicitor proteins orpolypeptides of Erwinia amylovora (see Kim et al., J. Bacteriol.180(19):5203-5210 (1998), which is hereby incorporated by reference inits entirety) or Pseudomonas syringae pv. tomato (see Charkowski et al.,J. Bacteriol. 180:5211-5217 (1998), which is hereby incorporated byreference in its entirety).

The protein or polypeptide of the present invention is preferablyproduced in purified form (preferably at least about 80%, morepreferably 90%, pure) by conventional techniques. Typically, the proteinor polypeptide of the present invention is secreted into the growthmedium of recombinant host cells. Such secretion can be performed inaccordance with the protocol established in PCT Application Publ. No. WO00/02996 to Bauer et al., which is hereby incorporated by reference inits entirety. Alternatively, the protein or polypeptide of the presentinvention is produced but not secreted into growth medium. In suchcases, to isolate the protein, the host cell (e.g., E. coli) carrying arecombinant plasmid is propagated, lysed by sonication, heat,differential pressure, or chemical treatment, and the homogenate iscentrifuged to remove bacterial debris. The supernatant is thensubjected to sequential ammonium sulfate precipitation. The fractioncontaining the polypeptide or protein of the present invention issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the proteins. If necessary, theprotein fraction may be further purified by high performance liquidchromatography (“HPLC”).

The DNA molecule encoding the hypersensitive response elicitorpolypeptide or protein can be incorporated in cells using conventionalrecombinant DNA technology. Generally, this involves inserting the DNAmolecule into an expression system to which the DNA molecule isheterologous (i.e., not normally present). The heterologous DNA moleculeis inserted into the expression system or vector in proper senseorientation and correct reading frame. The vector contains the necessaryelements for the transcription and translation of the insertedprotein-coding sequences. As discussed more fully below, the elementsfor transcription and translation differ depending on the type of systembeing employed (e.g., eukaryotic vs. prokaryotic, and plant vs. animal).

The present invention also relates to an expression vector containing aDNA molecule encoding a hypersensitive response elicitor protein. Thenucleic acid molecule of the present invention may be inserted into anyof the many available expression vectors using reagents that are wellknown in the art. In preparing a DNA vector for expression, the variousDNA sequences may normally be inserted or substituted into a bacterialplasmid. Any convenient plasmid may be employed, which will becharacterized by having a bacterial replication system, a marker whichallows for selection in a bacterium, and generally one or more unique,conveniently located restriction sites. Numerous plasmids, referred toas transformation vectors, are available for plant transformation. Theselection of a vector will depend on the preferred transformationtechnique and target species for transformation.

A variety of vectors are available for stable transformation usingAgrobacterium tumefaciens, a soilborne bacterium that causes crown gall.Crown gall is characterized by tumors or galls that develop on the lowerstem and main roots of the infected plant. These tumors are due to thetransfer and incorporation of part of the bacterium plasmid DNA into theplant chromosomal DNA. This transfer DNA (T-DNA) is expressed along withthe normal genes of the plant cell. The plasmid DNA, pTI, or Ti-DNA, for“tumor inducing plasmid,” contains the vir genes necessary for movementof the T-DNA into the plant chromosomal DNA. The T-DNA carries genesthat encode proteins involved in the biosynthesis of plant regulatoryfactors, and bacterial nutrients (opines). The T-DNA is delimited by two25 bp imperfect direct repeat sequences called the “border sequences.”By removing the oncogene and opine genes, and replacing them with a geneof interest, it is possible to transfer foreign DNA into the plantwithout the formation of tumors or the multiplication of Agrobacteriumtumefaciens. See Fraley et al., Proc. Nat'l Acad. Sci., 80:4803-4807(1983), which is hereby incorporated by reference in its entirety.

Other suitable vectors for practicing the present invention include, butare not limited to, the following viral vectors such as lambda vectorsystem gt11, gtWES.tB, Charon 4, and plasmid vectors such as pBR322,pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/− (see “StratageneCloning Systems” Catalog (1993) from LaJolla, Calif., which is herebyincorporated by reference in its entirety), pQE, pIH821, pGEX, pETseries (Studier et al., Methods in Enzymology. 185:60-89 (1990) which ishereby incorporated by reference in its entirety), and any derivativesthereof. Any appropriate vectors now known or later described forgenetic transformation are suitable for use with the present invention.Recombinant molecules can be introduced into cells via transformation,particularly transduction, conjugation, mobilization, orelectroporation. The DNA sequences are cloned into the vector usingstandard cloning procedures in the art as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, ColdSprings Harbor, N.Y. (1989), which is hereby incorporated by referencein its entirety.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference in its entirety, describes the production of expressionsystems in the form of recombinant plasmids using restriction enzymecleavage and ligation with DNA ligase. These recombinant plasmids arethen introduced by means of transformation and replicated in unicellularcultures including prokaryotic organisms and eukaryotic cells grown intissue culture.

Recombinant genes may also be introduced into viruses, such as vacciniavirus. Recombinant viruses can be generated by transfection of plasmidsinto cells infected with virus.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria. Theexpression elements of these vectors vary in their strength andspecificities. Depending upon the host-vector system utilized, any oneof a number of suitable transcription and translation elements can beused.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (mRNA)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eukaryotic promotersdiffer from those of prokaryotic promoters. Furthermore, eukaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a prokaryotic system, and, further, prokaryoticpromoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presenceof the proper prokaryotic signals which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference in its entirety.

Promoters vary in their “strength” (i.e., their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in prokaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7-9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the Ngene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

In one aspect of the present invention, the nucleic acid molecule of thepresent invention is incorporated into an appropriate vector in thesense direction, such that the open reading frame is properly orientedfor the expression of the encoded protein under control of a promoter ofchoice. This involves the inclusion of the appropriate regulatoryelements into the DNA-vector construct. These include non-translatedregions of the vector, useful promoters, and 5′ and 3′ untranslatedregions which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used.

A constitutive promoter is a promoter that directs expression of a genethroughout the development and life of an organism. Examples of someconstitutive promoters that are widely used for inducing expression oftransgenes include, without limitation, the nopoline synthase (NOS) genepromoter from Agrobacterium tumefaciens (U.S. Pat. No. 5,034,322 toRogers et al., which is hereby incorporated by reference in itsentirety), the cauliflower mosaic virus (CaMv) 35S and 19S promoters(U.S. Pat. No. 5,352,605 to Fraley et al., which is hereby incorporatedby reference in its entirety), those derived from any of the severalactin genes, which are known to be expressed in most cells types (U.S.Pat. No. 6,002,068 to Privalle et al., which is hereby incorporated byreference in its entirety), the ubiquitin promoter, which is a geneproduct known to accumulate in many cell types, the enhanced 35Spromoter described in U.S. Pat. No. 5,106,739 to Comai et al. (which ishereby incorporated by reference in its entirety), the dual 35Spromoter, the FMV promoter from figwort mosaic virus that is describedin U.S. Pat. No. 5,378,619 to Rogers et al. (which is herebyincorporated by reference in its entirety), the RIT-DNA promoterdescribed in U.S. Pat. No. 5,466,792 to Slightom et al. (which is herebyincorporated by reference in its entirety), the octopine T-DNA promoterdescribed in U.S. Pat. No. 5,428,147 to Barker et al. (which is herebyincorporated by reference in its entirety), the alcohol dehydrogenase 1promoter (Callis et al., Genes Dev., 1(10):1183-1200 (1987), which ishereby incorporated by reference in its entirety), the patatin promoterB33 (Rocha-Sosa et al., EMBO J., 8:23-29 (1989), which is herebyincorporated by reference in its entirety), the E8 promoter (Deikman etal., EMBO J., 7(11):3315-3320 (1988), which is hereby incorporated byreference in its entirety), the beta-conglycin promoter (Tierney et al.,Planta, 172:356-363 (1987), which is hereby incorporated by reference inits entirety), the acid chitinase promoter (Samac et al., PlantPhysiol., 93:907-914 (1990), which is hereby incorporated by referencein its entirety), the Arabidopsis histone H4 promoter described in U.S.Pat. No. 5,491,288 to Chaubet et al. (which is hereby incorporated byreference in its entirety), or the recombinant promoter for expressionof genes in monocots described in U.S. Pat. No. 5,290,924 to Last et al.(which is hereby incorporated by reference in its entirety).

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed. The inducer can be achemical agent, such as a metabolite, growth regulator, herbicide orphenolic compound, or a physiological stress directly imposed upon theplant such as cold, heat, salt, toxins, or through the action of apathogen or disease agent such as a virus or fungus. A plant cellcontaining an inducible promoter may be exposed to an inducer byexternally applying the inducer to the cell or plant such as byspraying, watering, heating, or by exposure to the operative pathogen.In addition, inducible promoters include promoters that function in atissue specific manner to regulate the gene of interest within selectedtissues of the plant Examples of such tissue specific promoters includeseed, flower, or root specific promoters as are well known in the field(U.S. Pat. No. 5,750,385 to Shewmaker et al., which is herebyincorporated by reference in its entirety).

In one aspect of the present invention, the inducible promoter is apathogen-inducible promoter. Such promoters include those frompathogenesis-related proteins (e.g., PR proteins, SAR proteins,beta-1,3-glucanase, chitinase), which are induced following infection bya pathogen (see, e.g., Redolfi et al., Neth. J. Plant Pathol. 89:245-254(1983); Uknes et al., Plant Cell 4:645-656 (1992); and Van Loon, PlantMol. Virol. 4:111-116 (1985), which are hereby incorporated by referencein their entirety). Another aspect of the present invention involvespromoters that are expressed locally at or near the site of pathogeninfection (see, e.g., Marineau et al., Plant Mol. Biol. 9:335-342(1987); Matton et al., Molecular Plant-Microbe Interactions 2:325-331(1989); Somsisch et al., Proc. Natl. Acad. Sci. U.S.A. 83:2427-2430(1986); Somsisch et al., Molecular and General Genetics 2:93-98 (1988);and Yang, Proc. Natl. Acad. Sci. U.S.A. 93:14972-14977 (1996), which arehereby incorporated by reference in their entirety). See also Chen etal., Plant J. 10:955-966 (1996); Zhang and Sing, Proc. Natl. Acad. Sci.U.S.A. 91:2507-2511 (1994); Warner et al., Plant J. 3:191-201 (1993);Siebertz et al., Plant Cell 1:961-968 (1989), which are herebyincorporated by reference in their entirety).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound inducible promoter may be used in the constructof the invention. Such wound inducible promoters include potatoproteinase inhibitor (pin II) gene (Ryan, Ann. Rev. Phytopath.28:425-449 (1990); Duan et al., Nature Biotechnology 14:494-498 (1996),which are hereby incorporated by reference in its entirety); wun1 andwun2, U.S. Pat. No. 5,428,148 to Reddy et al. (which is herebyincorporated by reference in its entirety); win1 and win2 (Stanford etal., Mol. Gen. Genet. 215:200-208 (1989), which is hereby incorporatedby reference in its entirety); systemin (McGurl et al., Science255:1570-1573 (1992), which is hereby incorporated by reference in itsentirety); WIP1 (Rohrmeier et al., Plant Mol Biol 22:783-792 (1993);Eckelkamp et al., FEBS Letters 323:73-76 (1993), which are herebyincorporated by reference in their entirety); and MPI gene (Cordero etal., Plant Journal 6(2): 141-150 (1994), which is hereby incorporated byreference in its entirety).

The DNA construct of the present invention also includes an operable 3′regulatory region, selected from among those which are capable ofproviding correct transcription termination and polyadenylation of mRNAfor expression in the host cell of choice, operably linked to a DNAmolecule which encodes for a protein of choice. A number of 3′regulatory regions are known to be operable in plants. Exemplary 3′regulatory regions include, without limitation, the nopaline synthase 3′regulatory region (Fraley et al., Proc. Nat'l Acad. Sci. USA80:4803-4807 (1983), which is hereby incorporated by reference in itsentirety) and the cauliflower mosaic virus 3′ regulatory region (Odellet al., Nature 313(6005):810-812 (1985), which is hereby incorporated byreference in its entirety). Virtually any 3′ regulatory region known tobe operable in plants would suffice for proper expression of the codingsequence of the DNA construct of the present invention.

The vector of choice, promoter, and an appropriate 3′ regulatory regioncan be ligated together to produce the DNA construct of the presentinvention using well known molecular cloning techniques as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, NY (1989), and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y.(1989), which are hereby incorporated by reference in their entirety.

Once the isolated DNA molecule encoding the hypersensitive responseelicitor polypeptide or protein has been cloned into an expressionsystem, it is ready to be incorporated into a host cell. Suchincorporation can be carried out by the various forms of transformationnoted above, depending upon the vector/host cell system. Suitable hostcells include, but are not limited to, bacteria, virus, yeast, mammaliancells, insect, plant, and the like.

The present invention further relates to methods of imparting diseaseresistance, stress resistance, and enhanced growth to plants or plantsgrown from treated seeds, controlling insects on plants or plants grownfrom treated plant seeds, imparting post-harvest disease or desiccationresistance in fruits or vegetables, imparting enhanced longevity offruit or vegetable ripeness, imparting desiccation resistance tocuttings of ornamental plants, and/or promoting early flowering ofornamental plants. These methods involve applying a hypersensitiveresponse elicitor polypeptide or protein in a non-infectious form to allor part of a plant, plant seed, cutting, and/or fruit or vegetable underconditions where the polypeptide or protein contacts all or part of thecells of the plant, plant seed, cutting, and/or fruit or vegetable.Alternatively, the hypersensitive response elicitor protein orpolypeptide can be applied to plants such that seeds recovered from suchplants themselves are able to impart disease resistance, stressresistance, and enhanced growth to plants grown from those seeds, tocontrol insects on plants grown from those seeds, to impart post-harvestdisease or desiccation resistance in fruits or vegetables harvested fromplants grown from those seeds, to impart enhanced longevity of fruit orvegetable ripeness for fruits or vegetables harvested from plants grownfrom those seeds, to impart desiccation resistance to cuttings ofornamental plants grown from those seeds, and/or promote early floweringof ornamental plants grown from those seeds.

Application of the hypersensitive response elicitor polypeptide orprotein in non-infectious form can be carried out in a number of waysincluding, without limitation: 1) application of an isolated elicitorpolypeptide or protein; 2) application of bacteria which do not causedisease and are transformed with genes encoding a hypersensitiveresponse elicitor polypeptide or protein; and 3) application of bacteriawhich cause disease in some plant species (but not in those to whichthey are applied) and naturally contain a gene encoding thehypersensitive response elicitor polypeptide or protein.

In one embodiment of the present invention, the hypersensitive responseelicitor polypeptide or protein of the present invention can be isolateddirectly from Pseudomonas syringae pv. tomato as described in theExamples infra. Preferably, however, the isolated hypersensitiveresponse elicitor polypeptide or protein of the present invention isproduced recombinantly and purified as described supra.

In other embodiments of the present invention, the hypersensitiveresponse elicitor polypeptide or protein of the present invention can beapplied to plants or plant seeds by applying bacteria containing genesencoding the hypersensitive response elicitor polypeptide or protein.Such bacteria must be capable of secreting or exporting the polypeptideor protein so that the elicitor can contact plant or plant seed cells.In these embodiments, the hypersensitive response elicitor polypeptideor protein is produced by the bacteria in planta or on seeds or justprior to introduction of the bacteria to the plants or plant seeds.

In one embodiment of the bacterial application mode of the presentinvention, the bacteria do not cause the disease and have beentransformed (e.g., recombinantly) with genes encoding a hypersensitiveresponse elicitor polypeptide or protein. For example, E. coli, whichdoes not elicit a hypersensitive response in plants, can be transformedwith genes encoding a hypersensitive response elicitor polypeptide orprotein and optionally type III secretion systems (U.S. patentapplication Ser. No. 09/350,852 to Bauer et al., filed Jul. 9, 1999,which is hereby incorporated by reference in its entirety), and thenapplied to plants. Bacterial species other than E. coli can also be usedin this embodiment of the present invention.

In another embodiment of the bacterial application mode of the presentinvention, the bacteria do cause disease and naturally contain a geneencoding a hypersensitive response elicitor polypeptide or protein.Examples of such bacteria are noted above. However, in this embodiment,these bacteria are applied to plants or their seeds which are notsusceptible to the disease carried by the bacteria For example,Pseudomonas syringae pv. tomato causes disease in tomato but not inbeans. However, such bacteria will elicit a hypersensitive response inbeans. Accordingly, in accordance with this embodiment of the presentinvention, Pseudomonas syringae pv. tomato can be applied to bean plantsor seeds to impart disease resistance to plants, enhance plant growth,and control insects on plants or plants grown from the plant seeds,impart stress resistance to plants, and/or impart post-harvest diseaseor desiccation resistance in fruits or vegetables, without causingdisease in that species.

The method of the present invention can be utilized to treat a widevariety of plants, their seeds, their cuttings, and/or their harvestedfruit or vegetables to impart disease resistance, stress resistance, andenhanced growth to plants or plants grown from treated seeds, to controlinsects on plants or plants grown from treated plant seeds, to impartpost-harvest disease or desiccation resistance in fruits or vegetables,to impart enhanced longevity of fruit or vegetable ripeness, to impartdesiccation resistance to cuttings of ornamental plants, and/or promoteearly flowering of ornamental plants.

Suitable plants include dicots and monocots. More particularly, usefulcrop plants can include, without limitation: alfalfa, rice, wheat,barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato,bean, pea, chicory, lettuce, endive, cabbage, brussel sprouts, beet,parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic,eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, tobacco, tomato, sorghum, and sugarcane. As used herein,ornamental plants are those plants that are not crop plants. Examples ofsuitable ornamental plants include, without limitation: Arabidopsisthaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum,carnation, rose, tulip, and zinnia.

With regard to the use of the hypersensitive response elicitor proteinor polypeptide of the present invention in imparting disease resistance,absolute immunity against infection may not be conferred, but theseverity of the disease is reduced and symptom development is delayed.Lesion number, lesion size, and extent of sporulation of fungalpathogens are all decreased. This method of imparting disease resistancehas the potential for treating previously untreatable diseases, treatingdiseases systemically which might not be treated separately due to cost,and avoiding the use of infectious agents or environmentally harmfulmaterials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi (see WO96/39802 to Wei et al. and WO 98/24297 to Qiu et al., which are herebyincorporated by reference in their entirety). Resistance, inter alia, tothe following viruses can be achieved by the method of the presentinvention: Tobacco mosaic virus and Tomato mosaic virus. Resistance,inter alia, to the following bacteria can also be imparted to plants inaccordance with present invention: Pseudomonas solancearum, Pseudomonassyringae pv. tabaci, and Xanthamonas campestris pv. pelargonii. Plantscan be made resistant, inter alia, to the following fungi by use of themethod of the present invention: Fusarium oxysporum and Phytophthorainfestans.

With regard to the use of the hypersensitive response elicitor proteinor polypeptide of the present invention to enhance plant growth, variousforms of plant growth enhancement or promotion can be achieved (WO98/32844 to Qiu et al., which is hereby incorporated by reference in itsentirety). This can occur as early as when plant growth begins fromseeds or later in the life of a plant. For example, plant growthaccording to the present invention encompasses greater yield, increasedquantity of seeds produced, increased percentage of seeds germinated,increased plant size, greater biomass, more and bigger fruit, earlierfruit coloration, and earlier fruit and plant maturation. As a result,the present invention provides significant economic benefit to growers.For example, early germination and early maturation permit crops to begrown in areas where short growing seasons would otherwise precludetheir growth in that locale. Increased percentage of seed germinationresults in improved crop stands and more efficient seed use. Greateryield, increased size, and enhanced biomass production allow greaterrevenue generation from a given plot of land.

Another aspect of the present invention is directed to effecting anyform of insect control for plants (WO 98/37752 to Zitter et al., whichis hereby incorporated by reference in its entirety). For example,insect control according to the present invention encompasses preventinginsects from contacting plants to which the hypersensitive responseelicitor has been applied, preventing direct insect damage to plants byfeeding injury, causing insects to depart from such plants, killinginsects proximate to such plants, interfering with insect larval feedingon such plants, preventing insects from colonizing host plants,preventing colonizing insects from releasing phytotoxins, etc. Thepresent invention also prevents subsequent disease damage to plantsresulting from insect infection.

The present invention is effective against a wide variety of insects.European corn borer is a major pest of corn (dent and sweet corn) butalso feeds on over 200 plant species including green, wax, and limabeans and edible soybeans, peppers, potato, and tomato plus many weedspecies. Additional insect larval feeding pests which damage a widevariety of vegetable crops include the following: beet armyworm, cabbagelooper, corn ear worm, fall armyworm, diamondback moth, cabbage rootmaggot, onion maggot, seed corn maggot, pickleworm (melonworm), peppermaggot, and tomato pinworm. Collectively, this group of insect pestsrepresents the most economically important group of pests for vegetableproduction worldwide.

The present invention relates to the use of the hypersensitive responseelicitors of the present invention to impart stress resistance to plants(WO 00/28055 to Wei et al., which is hereby incorporated by reference inits entirety). Resistance can be afforded to a variety of stresses, suchas any environmental factor having an adverse effect on plant physiologyand development. Examples of such environmental stress include withoutlimitation: climate-related stress (e.g., drought, water, frost, coldtemperature, high temperature, excessive light, and insufficient light),air pollution stress (e.g., carbon dioxide, carbon monoxide, sulfurdioxide, NO_(X), hydrocarbons, ozone, ultraviolet radiation, acidicrain), chemical (e.g., insecticides, fungicides, herbicides, heavymetals), and nutritional stress (e.g., fertilizer, micronutrients,macronutrients).

The present invention is also effective in imparting post-harvestdisease or desiccation resistance in fruits or vegetables (WO 01/80639to Wei et al., which is hereby incorporated by reference in itsentirety), as well as imparting desiccation resistance to cuttings ofornamental plants (WO 02/37960 to Wei et al., which is herebyincorporated by reference in its entirety). The present invention alsorelates to ornamental cuttings that have themselves been treated with ahypersensitive response elicitor protein or polypeptide of the presentinvention following removal from an ornamental plant, as well ascuttings removed from ornamental plants that have been treatedtherewith. The present invention also includes fruit or vegetableproducts that have been treated with a hypersensitive response elicitorprotein or polypeptide of the present invention or removed from plantsthat have been treated therewith.

In addition to imparting post-harvest disease or desiccation resistance,the longevity of fruit or vegetable ripeness can be enhanced (WO01/80639 to Wei et al., which is hereby incorporated by reference in itsentirety). Enhanced ripeness longevity will afford a longer shelf-lifeto produce and thereby promote less consumer waste.

The method of the present invention involving application of thehypersensitive response elicitor polypeptide or protein can be carriedout through a variety of procedures when all or part of the plant istreated, including leaves, stems, roots, propagules (e.g., cuttings),etc. This may (but need not) involve infiltration of the hypersensitiveresponse elicitor polypeptide or protein into the plant. Suitableapplication methods include high or low pressure spraying, injection,and leaf abrasion proximate to when elicitor application takes place.Seed treatments can also be employed, as described in U.S. Pat. No.6,235,974 to Qiu et al., which is hereby incorporated by reference inits entirety. When treating plant seeds, in accordance with theapplication embodiment of the present invention, the hypersensitiveresponse elicitor protein or polypeptide can be applied by low or highpressure spraying, coating, immersion, or injection (U.S. Pat. No.6,235,974 to Qiu et al., which is hereby incorporated by reference inits entirety). Other suitable application procedures can be envisionedby those skilled in the art provided they are able to effect contact ofthe hypersensitive response elicitor polypeptide or protein with cellsof the plant or plant seed. Once treated with the hypersensitiveresponse elicitor of the present invention, the seeds can be planted innatural or artificial soil and cultivated using conventional proceduresto produce plants. After plants have been propagated from seeds treatedin accordance with the present invention, the plants may be treated withone or more applications of the hypersensitive response elicitor proteinor polypeptide to impart disease resistance, stress resistance, andenhanced growth to plants or plants grown from treated seeds, to controlinsects on plants or plants grown from treated plant seeds, to impartpost-harvest disease or desiccation resistance in fruits or vegetables,to impart enhanced longevity of fruit or vegetable ripeness, to impartdesiccation resistance to cuttings of ornamental plants, and/or promoteearly flowering of ornamental plants.

The hypersensitive response elicitor polypeptide or protein can beapplied to plants or plant seeds in accordance with the presentinvention alone or in a mixture with other materials. Alternatively, thehypersensitive response elicitor polypeptide or protein can be appliedseparately to plants with other materials being applied at differenttimes.

A composition suitable for treating plants or plant seeds in accordancewith the application embodiment of the present invention contains ahypersensitive response elicitor polypeptide or protein in a carrier.Suitable carriers include water, aqueous solutions, slurries, or drypowders. By way of example, one such composition of harpin_(Ea) (3 wt %)is commercially available from Eden Bioscience Corp. under the tradenameMessenger®. It is expected that compositions of the proteins of thepresent invention can be prepared in a manner similar to that which isused for Messenger®.

Although not required, the composition of the present invention maycontain additional additives including fertilizer, insecticide,fungicide, nematacide, and mixtures thereof. Suitable fertilizersinclude (NH₄)₂NO₃. An example of a suitable insecticide is Malathion.Useful fungicides include Captan.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate the process of the present invention. In addition, thehypersensitive response elicitor polypeptide or protein can be appliedto plant seeds with other conventional seed formulation and treatmentmaterials, including clays and polysaccharides.

As an alternative to applying a hypersensitive response elicitorpolypeptide or protein to plants or plant seeds in order to impartdisease resistance, stress resistance, and enhanced growth to plants orplants grown from treated seeds, to control insects on plants or plantsgrown from treated plant seeds, to impart post-harvest disease ordesiccation resistance in fruits or vegetables, to impart enhancedlongevity of fruit or vegetable ripeness, to impart desiccationresistance to cuttings of ornamental plants, and/or promote earlyflowering of ornamental plants, transgenic plants or plant seeds can beutilized (WO 01/95724 to Wei et al., which is hereby incorporated byreference in its entirety). Using transgenic plants involves providing atransgenic plant transformed with a DNA molecule encoding ahypersensitive response elicitor polypeptide or protein of the presentinvention and growing the plant under conditions effective to permitthat DNA molecule to impart disease resistance, stress resistance, andenhanced growth to the transgenic plants, to control insects on thetransgenic plants, to impart post-harvest disease or desiccationresistance in fruits or vegetables removed from the transgenic plants,to impart enhanced longevity of fruit or vegetable ripeness, to impartdesiccation resistance to cuttings of transgenic ornamental plants,and/or promote early flowering of transgenic ornamental plants.Alternatively, a transgenic plant seed transformed with a DNA moleculeencoding a hypersensitive response elicitor polypeptide or protein ofthe present invention can be provided and planted in soil. A plant isthen propagated from the planted seed under conditions effective topermit that DNA molecule to impart disease resistance, stressresistance, and enhanced growth to plants grown from the transgenicseeds, to control insects on plants grown from the transgenic plantseeds, to impart post-harvest disease or desiccation resistance infruits or vegetables removed from the transgenic plants, to impartenhanced longevity of fruit or vegetable ripeness, to impart desiccationresistance to cuttings of transgenic ornamental plants, and/or promoteearly flowering of transgenic ornamental plants.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a hypersensitive responseelicitor polypeptide or protein need not but may be applied topically tothe transgenic plants or transgenic plant seeds.

The vector described above can be microinjected directly into plantcells by use of micropipettes to transfer mechanically the recombinantDNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is herebyincorporated by reference in its entirety. The genetic material may alsobe transferred into the plant cell using polyethylene glycol. Krens etal., Nature, 296:72-74 (1982), which is hereby incorporated by referencein its entirety.

Another approach to transforming plant cells with a gene which impartsresistance to pathogens is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference in their entirety. Generally, thisprocedure involves propelling inert or biologically active particles atthe cells under conditions effective to penetrate the outer surface ofthe cell and to be incorporated within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies. Fraley et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference in itsentirety.

The DNA molecule may also be introduced into the plant cells byelectroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference in its entirety. Inthis technique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C.

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. Its species are responsible for crown gall (A.tumefaciens) and hairy root disease (A. rhizogenes). The plant cells incrown gall tumors and hairy roots are induced to produce amino acidderivatives known as opines, which are catabolized only by the bacteria.The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome (Schell, Science, 237:1176-83 (1987), which is herebyincorporated by reference in its entirety).

After transformation, the transformed plant cells can be selected (usingappropriate selection media to identify transformants) and thenregenerated. Plant regeneration from cultured protoplasts is describedin Evans et al., Handbook of Plant Cell Cultures, Vol. 1: MacMillanPublishing Co., New York (1983); and Vasil I. R. (ed.), Cell Culture andSomatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984) andVol. III (1986), which are hereby incorporated by reference in theirentirety. It is known that practically all plants can be regeneratedfrom cultured cells or tissues.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfaEfficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedures. Transgenicseeds can, of course, be recovered from the transgenic plants. Theseseeds can then be planted in the soil and cultivated using conventionalprocedures to produce transgenic plants.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used topically to treat the plants and seeds. Theseother materials, including hypersensitive response elicitors, can beapplied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the hypersensitive response elicitor to impart diseaseresistance, stress resistance, and enhanced growth to plants, to controlinsects on plants, to impart post-harvest disease or desiccationresistance in fruits or vegetables, to impart enhanced longevity offruit or vegetable ripeness, to impart desiccation resistance tocuttings of ornamental plants, and/or promote early flowering ofornamental plants.

Such plants may also be treated with conventional plant treatment agents(e.g., insecticides, fertilizers, etc.). The present invention alsorelates to fruit or vegetables removed from transgenic plants of thepresent invention as well as cuttings removed from transgenic ornamentalplants of the present invention.

In the embodiment of the present invention where transgenic plants orplant seeds are utilized, it should be appreciated that the transgenicplants or plant seeds can include one or more transgenes other than thetransgene encoding the hypersensitive response elicitor protein orpolypeptide of the present invention. In particular, the use ofhypersensitive response elicitors for the purpose of maximizing thebenefit of a transgenic trait or overcoming a concomitant yield penaltyis disclosed in U.S. patent application Ser. No. 09/880,371 to Wei etal., filed Jun. 13, 2001 (now published), which is hereby incorporatedby reference in its entirety.

EXAMPLES

Each of the Examples set forth below is intended to illustrate thenature of the present invention but is by no means intended to limit itsscope.

Example 1 Isolation of HopPtoP and HopPmaH_(Pto) DNA Molecules andPreparation of Expression Vectors

-   -   hopPtoP was identified by using a reporter transposon to        identify genes in the P. s. tomato DC3000 genome that were        induced in a HrpL-dependent manner (Fouts et al., Proc. Natl.        Acad. Sci. USA 99(4):2275-2280 (2002) and supplemental materials        available online, which are hereby incorporated by reference in        their entirety). hopPmaH_(Pto) was identified by the        bioinformatic approach of scanning the DC3000 genome with a        Hidden Markov Model for Hrp promoter sequences (Fouts et al.,        Proc. Natl. Acad. Sci. USA 99(4):2275-2280 (2002) and        supplemental materials available online, which are hereby        incorporated by reference in their entirety). The HrpL-dependent        expression of hopPmaH_(Pto) was then confirmed by microarray        analysis.

The following primers were used to amplify hopPtoP and hopPmaH_(Pto)from P. syringae pv. tomato DC3000 genomic DNA using standard PCRprotocols:

hopPtoP Forward Primer caccatgacc atgggtgttt cac (SEQ ID NO:5) 23

hopPtoP Reverse Primer agcgggtaaa ttgccctgc (SEQ ID NO:6) 19

hopPmaH_(Pto) Forward Primer caccatgaat acgatcaac (SEQ ID NO:7) 19

hopPmaH_(Pto) Reverse Primer tttcacgacc tgtgc (SEQ ID NO:8) 15

Blunt end PCR fragments were generated for use with Invitrogen™ Gateway™technology in accordance with the manufacturer's instructions. PCRproducts were cloned into pENTR/SD/D-TOPO® vector using Gateway™technology. The Gateway™ technology offers a universal cloning processbased on the site-specific recombination properties of bacteriophagelambda, and provides a rapid and highly efficient way to move DNAsequences into multiple vector systems for functional analysis andprotein expression.

Briefly, the attachment sites for the vector can recombine with eachother. Thus, once an entry clone is created with the pENTR cloning kit,the fragment/gene of interest can easily be cloned into numerous othervectors that contain complementary attachment sites with which the pENTRattachment sites can recombine.

-   -   pENTR/SD/D-TOPO®:: hopPtoP and pENTR/SD/D-TOPO®:: hopPmaH_(Pto)        were used to clone into pET-DEST42, a ‘Gateway-ized’ vector from        Invitrogen™. This placed 6×-histidine and V5 epitope tags to the        C-terminal ends of hopPtoP and hopPmaH_(Pto). The resulting        plasmids were designated pCPP5098 (hopPtoP-6×His-V5) and        pCPP5099 (hopPmaH_(Pto)-6×His-V5). These plasmids were        transformed into E. coli BL21 DE3 from Novagen. QIAGEN protein        purification protocols were used to optimize purification under        native conditions. For HopPtoP, conditions described in Alfano        et al. (Molecular Microbiology 19:715-728 (1996), which is        hereby incorporated by reference in its entirety) were used for        purification and expression. The conditions described in Alfano        et al., Molecular Microbiology 19:715-728 (1996), which is        hereby incorporated by reference in its entirety, can also be        used for purification and expression of HopPmaH_(Pto). In        addition, standard conditions well known in the art for        growing E. coli (Sambrook et al., Molecular Cloning: A        Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY        (1989), which is hereby incorporated by reference in its        entirety), as well as for purifying His-tagged proteins        (Hainfeld et al., Microsc. Microanal. 8(Supp. 2):832CD (2002);        Hochuli et al., J. Chromatogr. 411:177-184 (1987); Hainfeld et        al., J. Struct. Biol. 127:185-198 (1999); Buchel et al., J. Mol.        Biol. 312:371-379 (2001); Hata et al., J. Virol. Methods        84(2):117-126 (2000); Blanc et al., J. Virol. Methods        77(1):11-15 (1999); and Schmidbauer et al., Biochemica 3:22-24        (1997), which are hereby incorporated by reference in their        entirety), can be used to purify and express HopPtoP and        HopPmaH_(Pto).

Example 2 Topical Application of the HopPtoP and HopPmaH_(Pto) to Plantsand HR Activity Thereof

Purified protein preparations prepared in accordance with Example 1above were used to infiltrate HopPtoP or HopPmaH_(Pto) onto planttissues. Purified HrpZ was similarly prepared. HrpZ, HopPtoP, andHopPmaH_(Pto) were purified using a 6×His tag system, and denatured at100° C. for 10 minutes. Proteins were then infiltrated into Nicotianatabacum cv. xanthi, and the hypersensitive response was photographed at24 hours (Alfano et al., Mol. Microbiol. 19:715-728 (1996), which ishereby incorporated by reference in its entirety). As shown in FIG. 2,like HrpZ, which is known to elicit a hypersensitive response in tobaccoplant tissues, HopPtoP and HopPmaH_(Pto) elicit a similar hypersensitiveresponse, while no such response could be detected from the negativecontrol.

Example 3 HopPtoP and HopPmaH_(Pto) Proteins are Secreted in Culture

pENTR/SD/D-TOPO®:: hopPtoP and pENTR/SD/D-TOPO®:: hopPmaH_(Pto) wereused to clone into pCPP3234, an IPTG-inducible “Gateway-ized” vectorcreated in the lab by cloning Gateway Reading Frame B fragment into SmaIsite of pCPP3214. pCPP3214 has a cyaa construct for gene fusions inbroad host range vector pVLT35 (de Lorenzo et al., Gene 123:17-24(1993), which is hereby incorporated by reference in its entirety) andwas constructed by digesting pMJH20 with SacI and HindIII, and ligatingthis fragment to pVLT35 cut with the same enzymes. This fused theadenylate cyclase (cyaA) gene to the C-terminal ends of hopPtoP andhopPmaH_(Pto), which provided an eptiope for detection of protein in thecell and supernatant fractions during the secretion assays. Theresulting plasmids, pCPP3256 (HopPtoP-CyaA) and pCPP3255(HopPmaH_(Pto)-CyaA), were then transformed into P. syringae DC3000 andCUCPB5114 (P. syringae DC3000 without the hrp/hrc cluster, negativecontrol).

Bacteria were inoculated in 30 mL Hrp minimal media (induces Hrp system)to OD₆₀₀=0.15 with IPTG to 100 uM. Bacteria were grown under standardconditions and then harvested at OD₆₀₀=0.3. Cultures were centrifuged at28000 rpm in Beckman L8-70 ultracentrifuge. The top 30 ml supernatantwas removed, 7.5 ml trichloroacetic acid (TCA) (˜25% TCA solution) wasadded to precipitate proteins, and the mixture was incubated at 4° C.overnight (>12 hours). Thereafter, supernatant fractions werecentrifuged at 28000 rpm in Beckman L8-70 ultracentrifuge to obtainsupernatant protein pellet, which was then washed twice with 5 ml 100%acetone. Protein was resuspended in 1× protein loading buffer, 100 μlfor every OD₆₀₀=0.3, and used 20 μl for SDS-PAGE analysis. Also, cellpellets (from above) were resuspended in 1 ml ddH₂O for every OD₆₀₀=0.3,and 15 μl was used for SDS-PAGE analysis. Standard SDS-PAGE analysis wasused.

CyaA antibodies (Santa Cruz Biotech; see Lee et al., Infect. Immun.67(5):2090-2095 (1999), which is hereby incorporated by reference in itsentirety) were used to detect HopPtoP and HopPmaH_(Pto) proteins in thecell and supernatant fractions. As shown in FIG. 3A, HopPtoP-CyaA fusionwas detected in subsequent Western analysis of the cell pellet (cell)and supernatant (SN) for P. syringae DC3000, which is known to possess atype III secretion system, but only in the cell pellet for P. syringaeDC3000 (Δhrp/hrc), which is lacking a type III secretion system. In FIG.3B, HopPmaH_(Pto)-CyaA fusion was detected in subsequent Westernanalysis of the cell pellet (cell) and supernatant (SN) for P. syringaeDC3000 but only in the cell pellet for P. syringae DC3000 (Δhrp/hrc).Thus, both HopPtoP and HopPmaH_(Pto) fusions were secreted by the typeIII secretion system of P. syringae of DC3000.

Example 4 HopPtoP and HopPmaH_(Pto) are Translocated into Plant Tissues

Vectors pCPP3256 (HopPtoP-CyaA) and pCPP3255 (HopPmaH_(Pto)-CyaA) in P.syringae DC3000, described above, and CUCPB5114 were used fortransloction assays. CyaA is the adenylate cyclase domain from thecyclolysin toxin from Bordetella pertussis. This domain uses calmodulin,found only in eukaryotic cells, to produce cAMP from ATP. Therefore,only a CyaA fusion protein that is translocated can produce high levelsof cAMP.

Translocation assays were performed by inoculating bacteria in 5 mM MES,pH=5.5 to OD₆₀₀=0.3, to which IPTG was added to 100 μM. The bacteriawere infiltrated into tomato cd. Moneymaker. 8 hours post-infiltration,one plant disc was taken from the tomato plant and then crushed withliquid nitrogen. To the crushed plant tissue, 300 μl 0.2M HCl was addedand the suspension was frozen at −20° C. The frozen samples wereanalyzed for cAMP levels with Correlate-EIA Direct cyclic AMP kit fromAssay Designs (see Petnicki-Ocwieja et al., Proc. Natl. Acad. Sci. USA99: 8336-8341 (2002) and accompanying supporting materials availableonline, which is hereby incorporated by reference in its entirety).

As shown in FIG. 4, HopPtoP and HopPmaH_(Pto), like the known harpinsHrpW and HrpZ, were translocated into tomato leaf tissue, as evidencedby the increased cAMP production by P. syringae DC3000 but not P.syringae DC3000 (Δhrp/hrc). In particular, HopPtoP is translocated atsignificantly higher levels than either HrpW or HrpZ.

Example 5 Transgenic Expression of HopPtoP and HopPmaH_(Pto) in Plants

A variety of technologies have been developed for production oftransgenic plants expressing HopPtoP and HopPmaH_(Pto). Each of thesetechnologies relies on the introduction of one of the recombinanthopPtoP and hopPmaH_(Pto) genes of the present invention into a plantcell or tissue and then regenerating the plant cell or tissue into atransgenic plant. The recombinant hopPtoP and hopPmaH_(Pto) genes cancontain either an inducible promoter or a constitutive promoter. Sincethe introduced gene is limited to a single function, other agronomicallyimportant traits of the crop plants remain unmodified. Using thistechnology, transgenic plants transformed with a recombinant hopPtoP orhopPmaH_(Pto) gene can be propagated and grown under conditionseffective to impart disease resistance, enhance plant growth, controlinsects, and impart stress resistance. Transgenic fruit- orvegetable-bearing plants can be useful for imparting post-harvestdisease or desiccation resistance in fruits or vegetables, whiletransgenic ornamental plants can be useful for imparting desiccationresistance to cuttings of ornamental plants and/or promoting earlyflowering of ornamental plants.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An isolated DNA molecule that encodes a hypersensitive responseeliciting protein or polypeptide and is selected from the groupconsisting of (a) a DNA molecule comprising the nucleotide sequence ofSEQ ID NO: 1 or SEQ ID NO: 3, (b) a DNA molecule encoding a protein orpolypeptide comprising the amino acid of SEQ ID NO: 2 or SEQ ID NO: 4,and (c) a DNA molecule that hybridizes to the complement of SEQ ID NO: 1or SEQ ID NO: 3 under hybridization conditions including a hybridizationmedium comprising 5×SSC buffer at a temperature of at least about 42° C.for about 18 hours; or an isolated DNA molecule that is complementary toat least one of DNA molecules (a), (b), or (c).
 2. The isolated DNAmolecule according to claim 1, wherein said DNA molecule comprises thenucleotide sequence of SEQ ID NO:
 1. 3. The isolated DNA moleculeaccording to claim 1, wherein said DNA molecule encodes a protein orpolypeptide comprising the amino acid of SEQ ID NO:
 2. 4. The isolatedDNA molecule according to claim 1, wherein said DNA molecule hybridizesto the complement of SEQ ID NO: 1 under hybridization conditionsincluding a hybridization medium comprising 5×SSC buffer at atemperature of at least about 42° C. for about 18 hours.
 5. The isolatedDNA molecule according to claim 1, wherein said DNA molecule comprisesthe nucleotide sequence of SEQ ID NO:
 3. 6. The isolated DNA moleculeaccording to claim 1, wherein said DNA molecule encodes a protein orpolypeptide comprising the amino acid of SEQ ID NO:
 4. 7. The isolatedDNA molecule according to claim 1, wherein said DNA molecule hybridizesto the complement of SEQ ID NO: 3 under hybridization conditionsincluding a hybridization medium comprising 5×SSC buffer at atemperature of at least about 42° C. for about 18 hours.
 8. The isolatedDNA molecule according to claim 1, wherein said DNA molecule iscomplementary to at least one of DNA molecules (a), (b), or (c).
 9. Anexpression vector comprising the DNA molecule of claim
 1. 10. Theexpression vector according to claim 9, wherein the DNA molecule is insense orientation and correct reading frame.
 11. A host cell transformedwith the DNA molecule of claim
 1. 12. The host cell according to claim11, wherein the host cell is selected from the group consisting of aplant cell and a bacterial cell.
 13. The host cell according to claim11, wherein the DNA molecule is present in an expression vector.
 14. Atransgenic plant transformed with the DNA molecule of claim
 1. 15. Thetransgenic plant according to claim 14, wherein the plant is selectedfrom the group consisting of alfalfa, rice, wheat, barley, rye, cotton,sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory,lettuce, endive, cabbage, brussel sprouts, beet, parsnip, turnip,cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper,celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon,citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco,tomato, sorghum, and sugarcane.
 16. The transgenic plant according toclaim 14, wherein the plant is selected from the group consisting ofArabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia,chrysanthemum, carnation, rose, tulip, and zinnia.
 17. A transgenicplant seed transformed with the DNA molecule of claim
 1. 18. Thetransgenic plant seed according to claim 17, wherein the plant seed isselected from the group consisting of alfalfa, rice, wheat, barley, rye,cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea,chicory, lettuce, endive, cabbage, brussel sprouts, beet, parsnip,turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant,pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple,pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean,tobacco, tomato, sorghum, and sugarcane seeds.
 19. The transgenic plantseed according to claim 17, wherein the plant seed is selected from thegroup consisting of Arabidopsis thaliana, Saintpaulia, petunia,pelargonium, poinsettia, chrysanthemum, carnation, rose, tulip, andzinnia seeds.
 20. An isolated hypersensitive response eliciting proteinor polypeptide selected from the group consisting of a protein orpolypeptide having the amino acid comprising SEQ ID NO: 2 or SEQ ID NO:4, and a protein or polyeptide encoded by a nucleic acid that hybridizesto the complement of SEQ ID NO: 1 or SEQ ID NO: 3 under hybridizationconditions including a hybridization medium comprising 5×SSC buffer at atemperature of at least about 42° C. for about 18 hours.
 21. Theisolated protein or polypeptide according to claim 20, wherein theprotein or polypeptide comprises the amino acid sequence of SEQ ID NO:2.
 22. The isolated protein or polypeptide according to claim 20,wherein the protein or polypeptide comprises the amino acid sequence ofSEQ ID NO:
 4. 23. The isolated protein or polypeptide according to claim20, wherein the protein or polypeptide is encoded by a nucleic acid thathybridizes to the complement of SEQ ID NO: 1 under hybridizationconditions including a hybridization medium comprising 5×SSC buffer at atemperature of at least about 42° C. for about 18 hours.
 24. Theisolated protein or polypeptide according to claim 20, wherein theprotein or polypeptide is encoded by a nucleic acid that hybridizes tothe complement of SEQ ID NO: 3 under hybridization conditions includinga hybridization medium comprising 5×SSC buffer at a temperature of atleast about 42° C. for about 18 hours.
 25. A composition comprising: aprotein or polypeptide according to claim 20 and a carrier.
 26. Acomposition according to claim 25 further comprising an additiveselected from the group consisting of fertilizer, insecticide,fungicide, nematacide, and mixtures thereof.
 27. A method of impartingdisease resistance to plants comprising: applying a protein orpolypeptide according to claim 20 in a non-infectious form to a plant orplant seed under conditions effective to impart disease resistance. 28.The method according to claim 27, wherein said applying is carried outwith a plant.
 29. The method according to claim 27, wherein saidapplying is carried out with a plant seed, said method furthercomprising: planting the seed treated with the hypersensitive responseelicitor in natural or artificial soil and propagating a plant from theseed planted in the soil.
 30. A method of enhancing plant growthcomprising: applying a protein or polypeptide according to claim 20 in anon-infectious form to a plant or plant seed under conditions effectiveto enhance plant growth.
 31. The method according to claim 30, whereinsaid applying is carried out with a plant.
 32. The method according toclaim 30, wherein said applying is carried out with a plant seed, saidmethod further comprising: planting the seed treated with thehypersensitive response elicitor in natural or artificial soil andpropagating a plant from the seed planted in the soil.
 33. A method ofinsect control for plants comprising: applying a protein or polypeptideaccording to claim 20 in a non-infectious form to a plant or plant seedunder conditions effective to control insects.
 34. The method accordingto claim 33, wherein said applying is carried out with a plant.
 35. Themethod according to claim 33, wherein said applying is carried out witha plant seed, said method further comprising: planting the seed treatedwith the hypersensitive response elicitor in natural or artificial soiland propagating a plant from the seed planted in the soil.
 36. A methodof imparting post-harvest disease resistance or post-harvest desiccationresistance to a fruit or vegetable comprising: applying a protein orpolypeptide according to claim 20 in a non-infectious form to a fruit orvegetable, a plant, or a plant seed under conditions effective to impartpost-harvest disease resistance or desiccation resistance to the fruitor vegetable.
 37. The method according to claim 36, wherein saidapplying is carried out with a fruit or vegetable.
 38. The methodaccording to claim 36, wherein said applying is carried out with aplant.
 39. The method according to claim 36, wherein said applying iscarried out with a plant seed, said method further comprising: plantingthe seed treated with the hypersensitive response elicitor in natural orartificial soil and propagating a plant from the seed planted in thesoil.
 40. A method of imparting desiccation resistance to cuttingsremoved from ornamental plants comprising: applying a protein orpolypeptide according to claim 20 in a non-infectious form to anornamental plant or plant seed, or a cutting removed therefrom, underconditions effective to impart desiccation resistance to cuttingsremoved from the ornamental plant.
 41. The method according to claim 40,wherein said applying is carried out with an ornamental plant.
 42. Themethod according to claim 40, wherein said applying is carried out witha cutting removed from an ornamental plant.
 43. The method according toclaim 40, wherein said applying is carried out with an ornamental plantseed, said method further comprising: planting the seed treated with thehypersensitive response elicitor in natural or artificial soil andpropagating a plant from the seed planted in the soil.
 44. A method ofpromoting early flowering of ornamental plants comprising: applying aprotein or polypeptide according to claim 20 in a non-infectious form toan ornamental plant or plant seed under conditions effective to promoteflowering of the treated ornamental plants, or plants grown from treatedseeds, that occurs earlier as compared to untreated ornamental plants.45. The method according to claim 44, wherein said applying is carriedout with an ornamental plant.
 46. The method according to claim 44,wherein said applying is carried out with an ornamental plant seed, saidmethod further comprising: planting the seed treated with thehypersensitive response elicitor in natural or artificial soil andpropagating a plant from the seed planted in the soil.
 47. A method ofenhancing the longevity of fruit or vegetable ripeness comprising:applying a protein or polypeptide according to claim 20 in anon-infectious form to a fruit or vegetable, a plant, or a plant seedunder conditions effective to enhance the longevity of fruit orvegetable ripeness.
 48. The method according to claim 47, wherein saidapplying is carried out with a fruit or vegetable.
 49. The methodaccording to claim 47, wherein said applying is carried out with aplant.
 50. The method according to claim 47, wherein said applying iscarried out with a plant seed, said method further comprising: plantingthe seed treated with the hypersensitive response elicitor in natural orartificial soil and propagating a plant from the seed planted in thesoil.
 51. A method of imparting stress resistance to plants comprising:applying a protein or polypeptide according to claim 20 in anon-infectious form to a plant or plant seed under conditions effectiveto impart stress resistance.
 52. The method according to claim 51,wherein said applying is carried out with a plant.
 53. The methodaccording to claim 51, wherein said applying is carried out with a plantseed, said method further comprising: planting the seed treated with thehypersensitive response elicitor in natural or artificial soil andpropagating a plant from the seed planted in the soil.
 54. A method ofimparting disease resistance to plants comprising: providing atransgenic plant or plant seed transformed with a DNA molecule accordingto claim 1 and growing the transgenic plant or transgenic plant producedfrom the transgenic plant seed under conditions effective to impartdisease resistance.
 55. The method according to claim 54, wherein atransgenic plant is provided.
 56. The method according to claim 54,wherein a transgenic plant seed is provided.
 57. A method of enhancingplant growth comprising: providing a transgenic plant or plant seedtransformed with a DNA molecule according to claim 1 and growing thetransgenic plant or transgenic plant produced from the transgenic plantseed under conditions effective to enhance plant growth.
 58. The methodaccording to claim 57, wherein a transgenic plant is provided.
 59. Themethod according to claim 57, wherein a transgenic plant seed isprovided.
 60. A method of insect control for plants comprising:providing a transgenic plant or plant seed transformed with a DNAmolecule according to claim 1 and growing the transgenic plant ortransgenic plant produced from the transgenic plant seed underconditions effective to control insects.
 61. The method according toclaim 60, wherein a transgenic plant is provided.
 62. The methodaccording to claim 60, wherein a transgenic plant seed is provided. 63.A method of imparting post-harvest disease resistance or desiccationresistance to a fruit or vegetable comprising: providing a transgenicfruit or vegetable plant or plant seed transformed with a DNA moleculeaccording to claim 1 and growing the transgenic plant or transgenicplant produced from the transgenic plant seed under conditions effectiveto impart post-harvest disease resistance or desiccation resistance tofruits or vegetables removed from the transgenic plant.
 64. The methodaccording to claim 63, wherein a transgenic plant is provided.
 65. Themethod according to claim 63, wherein a transgenic plant seed isprovided.
 66. A method of enhancing the longevity of fruit or vegetableripeness comprising: providing a transgenic fruit or vegetable plant orplant seed transformed with a DNA molecule according to claim 1 andgrowing the transgenic plant or transgenic plant produced from thetransgenic plant seed under conditions effective to enhance thelongevity of ripeness for fruits or vegetables harvested therefrom. 67.The method according to claim 66, wherein a transgenic plant isprovided.
 68. The method according to claim 66, wherein a transgenicplant seed is provided.
 69. A method of imparting desiccation resistanceto ornamental plant cuttings comprising: providing a transgenicornamental plant or plant seed transformed with a DNA molecule accordingto claim 1 and growing the transgenic plant or transgenic plant producedfrom the transgenic plant seed under conditions effective to impartdesiccation resistance to a cutting from the transgenic plant.
 70. Themethod according to claim 69, wherein a transgenic plant is provided.71. The method according to claim 69, wherein a transgenic plant seed isprovided.
 72. A method of promoting early flowering of ornamental plantscomprising: providing a transgenic ornamental plant or plant seedtransformed with a DNA molecule according to claim 1 and growing thetransgenic plant or transgenic plant produced from the transgenic plantseed under conditions effective to promote early flowering in thetransgenic plant.
 73. The method according to claim 72, wherein atransgenic plant is provided.
 74. The method according to claim 72,wherein a transgenic plant seed is provided.
 75. A method of impartingstress resistance to plants comprising: providing a transgenic plant orplant seed transformed with a DNA molecule according to claim 1 andgrowing the transgenic plant or transgenic plant produced from thetransgenic plant seed under conditions effective to impart stressresistance to the transgenic plant.
 76. The method according to claim75, wherein a transgenic plant is provided.
 77. The method according toclaim 75, wherein a transgenic plant seed is provided.
 78. A method ofimparting desiccation resistance to cuttings removed from ornamentalplants comprising: providing a transgenic ornamental plant or plant seedtransformed with a DNA molecule according to claim 1 and growing thetransgenic plant or transgenic plant produced from the transgenic plantseed under conditions effective to impart desiccation resistance tocuttings removed from the ornamental plant.
 79. The method according toclaim 78, wherein a transgenic plant is provided.
 80. The methodaccording to claim 78, wherein a transgenic plant seed is provided.